Glycoprotein B (gB) of pseudorabies virus (PrV) is essential for virus entry into target cells and direct viral cell-to-cell spread. Recently, we described a carboxy-terminally truncated derivative of PrV gB, gB-007, which was inefficiently incorporated into virions, was unable to complement infectivity, but was fully capable of restoring direct viral cell-to-cell spread of gB-negative PrV (R. Nixdorf, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 74:7137-7145, 2000). Since recombinant PrV-007, which expresses gB-007 instead of wild-type gB, was able to spread directly from cell to cell, we attempted to obtain compensatory mutations leading to restoration of the entry defect by performing serial passages in cell culture. This procedure has previously been used to successfully restore entry defects in gD- or gL-deficient PrV mutants. From an initial titer of 100 PFU per ml in the supernatant, titers increased, reaching wild-type levels of up to 10⁷ PFU after ca. 20 passages. One single-plaque isolate of the passaged mutant, designated PrV-007Pass, was further characterized. PrV-007Pass gB was efficiently incorporated into the viral envelope and restored infectivity to a gB-negative PrV mutant, PrV-gB. Interestingly, localization of PrV-007Pass gB in the plasma membrane was similar to that of PrV-007. In contrast, wild-type gB is mainly found in intracellular vesicles. Marker rescue experiments and trans-complementation assays demonstrated the presence of compensatory mutations within the gB gene of PrV-007Pass. DNA sequencing revealed two point mutations in the gB open reading frame of PrV-007Pass, resulting in amino acid substitutions at positions 305 and 744 of gB, both of which are required for compensation of the defect in PrV-007. Our data again demonstrate the power of reversion analysis of herpesviruses and suggest that cytosolic and ectodomains play a role in incorporation of gB into virions.